Nucleic acid-containing lipid particles and related methods
Lipid particles containing a nucleic acid, devices and methods for making the lipid particles, and methods for using the lipid particles.
1. A method for making lipid particles with encapsulated nucleic acid, comprising:
(a) introducing a first stream comprising a nucleic acid in a first solvent into a microfluidic device; wherein the device has a first region adapted for flowing one or more streams introduced into the device and a second region for mixing the contents of the one or more streams with a microfluidic mixer;
(b) introducing a second stream comprising lipid particle-forming materials in a second solvent into the device to provide first and second streams flowing under laminar flow conditions, wherein the lipid particle-forming materials comprise a cationic lipid, and wherein the first and second solvents are not the same;
(c) flowing the one or more first streams and the one or more second streams from the first region of the device into the second region of the device; and
(d) mixing the one or more first streams and the one or more second streams in the second region of the device to provide a third stream comprising lipid particles with encapsulated nucleic acid,
wherein the lipid particles with encapsulated nucleic acid comprise:
(i) a substantially solid core consisting of nucleic acid, cationic lipid, and optionally second lipids; and
(ii) PEG-lipid surrounding the core,
wherein the lipid particle has a core and a periphery, the core has an area-averaged electron density that is not less than 20% of the maximum density of the periphery as measured by cryo transmission electron microscopy, and
wherein the 31 P nuclear magnetic resonance spectrum of the lipid particle measured in solution after treatment with 150 mM ammonium acetate does not exhibit a resonance due to the nucleic acid.
2. The method of claim 1 , wherein the nucleic acid is encapsulated in the lipid particle with an efficiency of from 90 to 100%.
3. The method of claim 1 , wherein the first solvent is an aqueous buffer and the second solvent is an aqueous alcohol.
4. The method of claim 1 , wherein the first solvent is an aqueous solvent and the second solvent is an organic solvent, and wherein the volume ratio between the first and second solvent exceeds 1.0 (aqueous:organic).
5. The method of claim 1 , wherein the one or more first streams and the one or more second streams are flowed at a flow rate from about 1 mL/min to about 40 mL/min.
6. The method of claim 1 , wherein the particle has a diameter from about 15 nm to about 300 nm.
7. The method of claim 1 , wherein the particle comprises from 30 to 95 mole percent cationic lipid.
8. The method of claim 1 , wherein the cationic lipid is an amino lipid.
9. The method of claim 1 , wherein the cationic lipid is selected from the group consisting of DODAC, DOTMA, DDAB, DOTAP, DOTAP⋅Cl, DC-Chol, DOSPA, DOGS, DOPE, DODAP, DODMA, and DMRIE.
10. The method of claim 1 , wherein the cationic lipid has the formula:
wherein R 1 and R 2 are either the same or different and independently optionally substituted C 10 -C 24 alkyl, optionally substituted C 10 -C 24 alkenyl, optionally substituted C 10 -C 24 alkynyl, or optionally substituted C 10 -C 24 acyl;
R 3 and R 4 are either the same or different and independently optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl, or optionally substituted C 2 -C 6 alkynyl or R 3 and R 4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;
R 5 is either absent or present and when present is hydrogen or C 1 -C 6 alkyl;
m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0;
q is 0, 1, 2, 3, or 4; and
Y and Z are either the same or different and independently O, S, or NH.
11. The method of claim 1 , comprising from about 1 to about 5 mole percent PEG-lipid.
12. The method of claim 1 , wherein the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols.
13. The method of claim 1 , wherein the PEG-lipid is selected from the group consisting of PEG-c-DOMG, PEG-c-DMA, and PEG-c-DMG.
14. The method of claim 1 , wherein the second lipids are selected from the group consisting of zwitterionic lipids, sterols, and mixtures thereof.
15. The method of claim 1 , wherein the second lipids are selected from the group consisting of diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.
16. The method of claim 1 , wherein the second lipid is cholesterol.
17. The method of claim 1 , wherein the nucleic acid is a DNA, an RNA, a locked nucleic acid, a nucleic acid analog, or a plasmid capable of expressing a DNA or an RNA.
18. The method of claim 1 , wherein the nucleic acid is ssDNA or dsDNA.
19. The method of claim 1 , wherein the nucleic acid is mRNA, siRNA, or microRNA.
20. The method of claim 1 , wherein the nucleic acid is an antisense oligonucleotide.